Development and Validation of Nerve-Targeted Bacteriochlorin Sensors.

We report an innovative approach to producing bacteriochlorins (bacs) via formal cycloaddition by subjecting a porphyrin to a trimolecular reaction. Bacs are near-infrared probes with the intrinsic ability to serve in multimodal imaging. However, despite their ability to fluoresce and chelate metal ions, existing bacs have thus offered limited ability to label biomolecules for target specificity or have lacked chemical purity, limiting their use in bio-imaging. In this work, bacs allowed a precise and controlled appending of clickable linkers, lending the porphyrinoids substantially more chemical stability, clickability, and solubility, rendering them more suitable for preclinical investigation. Our bac probes enable the targeted use of biomolecules in fluorescence imaging and Cerenkov luminescence for guided intraoperative imaging. Bacs' capacity for chelation provides opportunities for use in non-invasive positron emission tomography/computed tomography. Herein, we report the labeling of bacs with Hs1a, a (NaV1.7)-sodium-channel-binding peptide derived from the Chinese tarantula Cyriopagopus schmidti to yield Bac-Hs1a and radiolabeled Hs1a, which shuttles our bac sensor(s) to mouse nerves. In vivo, the bac sensor allowed us to observe high signal-to-background ratios in the nerves of animals injected with fluorescent Bac-Hs1a and radiolabeled Hs1a in all imaging modes. This study demonstrates that Bac-Hs1a and [64Cu]Cu-Bac-Hs1a accumulate in peripheral nerves, providing contrast and utility in the preclinical space. For the chemistry and bio-imaging fields, this study represents an exciting starting point for the modular manipulation of bacs, their development and use as probes for diagnosis, and their deployment as formidable multiplex nerve-imaging agents for use in routine imaging experiments.

Bimodal Imaging of Mouse Peripheral Nerves with Chlorin Tracers.

Almost 17 million Americans have a history of cancer, a number expected to reach over 22 million by 2030. Cancer patients often undergo chemotherapy in the form of antineoplastic agents such as cis-platin and paclitaxel. Though effective, these agents can induce debilitating side effects; the most common neurotoxic effect, chemotherapy-induced peripheral neuropathy (CIPN), can endure long after treatment ends. Despite the widespread and chronic nature of the dysfunction, no tools exist to quantitatively measure chemotherapy-induced peripheral neuropathy. Such a tool would not only benefit patients but their stratification could also save significant financial and social costs associated with neuropathic pain. In our first step toward addressing this unmet clinical need, we explored a novel dual approach to localize peripheral nerves: Cerenkov luminescence imaging (CLI) and fluorescence imaging (FI). Our approach revolves around the targeting and imaging of voltage-gated sodium channel subtype NaV1.7, highly expressed in peripheral nerves from both harvested human and mouse tissues. For the first time, we show that Hsp1a, a radiolabeled NaV1.7-selective peptide isolated from Homoeomma spec. Peru, can serve as a targeted vector for delivering a radioactive sensor to the peripheral nervous system. In situ, we observe high signal-to-noise ratios in the sciatic nerves of animals injected with fluorescently labeled Hsp1a and radiolabeled Hsp1a. Moreover, confocal microscopy on fresh nerve tissue shows the same high ratios of fluorescence, corroborating our in vivo results. This study indicates that fluorescently labeled and radiolabeled Hsp1a tracers could be used to identify and demarcate nerves in a clinical setting.

Facile Preparation of Drug-Loaded Tristearin Encapsulated Superparamagnetic Iron Oxide Nanoparticles Using Coaxial Electrospray Processing.

Naturally occurring polymers are promising biocompatible materials that have many applications for emerging therapies, drug delivery systems, and diagnostic agents. The handling and processing of such materials still constitutes a major challenge, which can limit the full exploitation of their properties. This study explores an ambient environment processing technique: coaxial electrospray (CO-ES) to encapsulate genistein (an isoflavonoid and model drug), superparamagnetic iron oxide nanoparticles (SPIONs, 10-15 nm), and a fluorophore (BODIPY) into a layered (triglyceride tristearin shell) particulate system, with a view to constructing a theranostic agent. Mode mapping of CO-ES led to an optimized atomization engineering window for stable jetting, leading to encapsulation of SPIONs within particles of diameter 0.65-1.2 µm and drug encapsulation efficiencies of around 92%. Electron microscopy was used to image the encapsulated SPIONs and confirm core-shell triglyceride encapsulation in addition to further physicochemical characterization (AFM, FTIR, DSC, and TGA). Cell viability assays (MTT, HeLa cells) were used to determine optimal SPION loaded particles (∼1 mg/mL), while in vitro release profile experiments (PBS, pH = 7.4) demonstrate a triphasic release profile. Further cell studies confirmed cell uptake and internalization at selected time points (t = 1, 2, and 4 h). The results suggest potential for using the CO-ES technique as an efficient way to encapsulate SPIONs together with sensitive drugs for the development of multimodal particles that have potential application for combined imaging and therapy.

Two novel ternary dicopper(II) µ-guanazole complexes with aromatic amines strongly activated by quantum dots for DNA cleavage.

Two novel (µ-guanazole)-bridged binuclear copper(II) complexes with 1,10-phenanthroline (phen) or 2,2'-bipyridine (bipy), [Cu2(µ-N2,N4-Hdatrz)(phen)2(H2O)(NO3)4] (1) and [Cu2(µ-N1,N2-datrz)2(µ-OH2)(bipy)2](ClO4)2 (2) (Hdatrz = 3,5-diamino-1,2,4-triazole = guanazole), have been prepared and characterized by X-ray diffraction, spectroscopy, and susceptibility measurements. Compounds 1 and 2 differ in the aromatic amine, which acts as a coligand, and in the Cu···Cu'-bridging system. Compound 1, which contains two mono-bridged copper ions, represents the first example of a discrete Cu-(NCN-trz)-Cu' complex. Compound 2, with two triply bridged copper ions, is one of the few compounds featuring a Cu-[(NN-trz)2 + (O-aquo)]-Cu' unit. Both compounds display antiferromagnetic coupling but of different magnitude: J (µ2,4-triazole) = -52 cm(-1) for 1 and J (µ1,2-triazolate) = -115 cm(-1) for 2. The DNA binding and cleavage properties of the two compounds have been investigated. Fluorescence, viscosimetry, and thermal denaturation studies reveal that both complexes have high affinity for DNA (1 > 2) and that only 1 acts as an intercalator. In the presence of a reducing agent like 3-mercaptopropionic acid, 1 produces significant oxidative DNA cleavage, whereas 2 is inactive. However, in the presence of very small quantities of micelles filled with core-shell CdSe-ZnS quantum dots (15 nM), 1 and 2 are considerably more active and become highly efficient nucleases as a result of the different possible mechanisms for promoting cooperative catalysis (metal-metal, metal-hydrogen bonding, metal-intercalation, and metal-nanoparticle). Electrophoresis DNA-cleavage inhibition experiments, X-ray photoelectron spectroscopy studies, and fluorescence ethidium bromide displacement assays reveal that in these novel nucleases the QDs act as redox-active protein-like nanoparticle structures that bind to the DNA and deliver electrons to the copper(II) centers for the generation of Cu(I) and reactive oxygen species.

Environmentally sensitive fluorescence of the topical retinoid adapalene.

Intrinsic fluorescence of drugs brings valuable information on their localization in the organism and their interaction with key biomolecules. In this work, we investigate the absorption and emission properties of the topical retinoid adapalene in different solvents and biological media. While the UVA/UVB absorption band does not exhibit any significant solvent-dependent behavior, a strong positive solvatochromism is observed for the emission. These results are in line with molecular modeling and simulations that show the presence of two quasi-degenerate states, i.e., a local π-π* and an intermolecular charge-transfer (ICT) state. However, molecular modeling also revealed that, whatever the solvent, at the corresponding equilibrium geometry the lowest and emissive excited state is the local π-π*. Finally, the potential of adapalene to act as a biological probe is demonstrated using albumin, DNA and micelles.

CXCR4: From Signaling to Clinical Applications in Neuroendocrine Neoplasms.

There are several well-described molecular mechanisms that influence cell growth and are related to the development of cancer. Chemokines constitute a fundamental element that is not only involved in local growth but also affects angiogenesis, tumor spread, and metastatic disease. Among them, the C-X-C motif chemokine ligand 12 (CXCL12) and its specific receptor the chemokine C-X-C motif receptor 4 (CXCR4) have been widely studied. The overexpression in cell membranes of CXCR4 has been shown to be associated with the development of different kinds of histological malignancies, such as adenocarcinomas, epidermoid carcinomas, mesenchymal tumors, or neuroendocrine neoplasms (NENs). The molecular synapsis between CXCL12 and CXCR4 leads to the interaction of G proteins and the activation of different intracellular signaling pathways in both gastroenteropancreatic (GEP) and bronchopulmonary (BP) NENs, conferring greater capacity for locoregional aggressiveness, the epithelial-mesenchymal transition (EMT), and the appearance of metastases. Therefore, it has been hypothesized as to how to design tools that target this receptor. The aim of this review is to focus on current knowledge of the relationship between CXCR4 and NENs, with a special emphasis on diagnostic and therapeutic molecular targets.

N-(5-Amino-1H-1,2,4-triazol-3-yl)pyridine-2-carboxamide.

The title compound, C(8)H(8)N(6)O, was obtained by the reaction of 3,5-diamino-1,2,4-triazole with ethyl 2-picolinate in a glass oven. The dihedral angles formed between the plane of the amide group and the pyridine and triazole rings are 11.8 (3) and 5.8 (3)°, respectively. In the crystal, an extensive system of classical N-H⋯N and N-H⋯O hydrogen bonds generate an infinite three-dimensional network.

A unique discrete tetranuclear Cu'-Cu(N-N)2Cu-Cu' copper(II) complex, built from a µ3-1,2,4-triazolato-µ-carboxylato ligand, as an effective DNA cleavage agent.

The title compound, characterized by means of an X-ray structure analysis, represents an easy example of a noncatena "1 + 2 + 1" tetranuclear copper(II) µ(3)-triazolate compound. [Cu(4)(atc)(2)(dien)(4)(ClO(4))(2)](ClO(4))(2)·2H(2)O (1), where H(2)atc = 5-amino-l,2,4-triazole-3-carboxylic acid and dien = diethylenetriamine = 1,4,7-triazaheptane, contains two copper atoms linked by a double diazinic bridge, each of which is further connected to a third and fourth copper atom (Cu') through the triply bridging triazolato ring and the bidentate carboxylato group of the atc(2-) ligands. The copper-copper distances within the tetranuclear unit are Cu-Cu = 4.059 Å, Cu-Cu' = 5.686 and 6.370 Å, and Cu'-Cu' = 11.373 Å. The compound self-assembles into a tridimensional hydrogen-bonded network to generate a MOF. 1 exhibits antiferromagnetic behavior with g = 2.10(1), J = -34.1(2) cm(-1) and j = -5.50(3) cm(-1), where J is the coupling constant of the central Cu-Cu pair and j the coupling constant of the two Cu-Cu' (Cu(central)-Cu(pheripheral)) pairs, as defined by H = -JS(2)S(2a) - j (S(1)S(2) + S(2a)S(1a)). Complex 1 has been tested as nuclease mimic. It shows good binding propensity to calf thymus DNA, with a binding constant value of 6.20 × 10(6) M(-1) (K(app)) and ΔT(m) = 18.3 °C. Moreover, the compound displays efficient oxidative cleavage of pUC18 DNA, even at low concentration, in the presence of a mild reducing agent (ascorbate), with a rate constant for the conversion of supercoiled to nicked DNA (k(obs)) of ~0.126 min(-1). The good reactivity of 1 toward DNA is explained from the electrostatic interactions of the cationic species produced in solution.

Recent Advances in Radiometals for Combined Imaging and Therapy in Cancer.

Nuclear medicine is defined as the use of radionuclides for diagnostic and therapeutic applications. The imaging modalities positron emission tomography (PET) and single-photon emission computed tomography (SPECT) are based on γ-emissions of specific energies. The therapeutic technologies are based on ß- -particle-, α-particle-, and Auger electron emitters. In oncology, PET and SPECT are used to detect cancer lesions, to determine dosimetry, and to monitor therapy effectiveness. In contrast, radiotherapy is designed to irreparably damage tumor cells in order to eradicate or control the disease's progression. Radiometals are being explored for the development of diagnostic and therapeutic radiopharmaceuticals. Strategies that combine both modalities (diagnostic and therapeutic), referred to as theranostics, are promising candidates for clinical applications. This review provides an overview of the basic concepts behind therapeutic and diagnostic radiopharmaceuticals and their significance in contemporary oncology. Select radiometals that significantly impact current and upcoming cancer treatment strategies are grouped as clinically suitable theranostics pairs. The most important physical and chemical properties are discussed. Standard production methods and current radionuclide availability are provided to indicate whether a cost-efficient use in a clinical routine is feasible. Recent preclinical and clinical developments and outline perspectives for the radiometals are highlighted in each section.

A kit-based aluminium-[18F]fluoride approach to radiolabelled microbubbles.

The production of 18F-labelled microbubbles (MBs) via the aluminium-[18F]fluoride ([18F]AlF) radiolabelling method and facile inverse-electron-demand Diels-Alder (IEDDA) 'click' chemistry is reported. An [18F]AlF-NODA-labelled tetrazine was synthesised in excellent radiochemical yield (>95% RCY) and efficiently conjugated to a trans-cyclooctene (TCO) functionalised phospholipid (40-50% RCY), which was incorporated into MBs (40-50% RCY). To demonstrate the potential of producing 18F-labelled MBs for clinical studies, we also describe a kit-based approach which is amenable for use in a hospital radiopharmacy setting.

Leveraging synthetic chlorins for bio-imaging applications.

Synthetic chlorins are not only fluorescent, the modulation of the tetrapyrrole system can also chelate metal ions. Conjugation of linkers at their pyrrolidines allows for conjugation to bio-molecules to create target specificity. By altering these chemo-photophysical properties, this work facilitates the use of chlorins in fluorescent imaging and positron emission tomography (PET).

Hexanuclear Cu3O-3Cu triazole-based units as novel core motifs for high nuclearity copper(ii) frameworks.

The asymmetric 3,5-disubstituted 1,2,4-triazole ligand H2V (5-amino-3-picolinamido-1,2,4-triazole) by reaction with an excess of Cu(ii) perchlorate (Cu : H2V being 12 : 1) has produced a novel hexanuclear {Cu6(µ3-O/H)(HV/V)3} fragment, with one triangular Cu3(µ3-O/H) group connected to three peripheral single Cu(ii) ions through a cis-cis-trans bridging mode of the ligand, which is the building block of the three structures described here: one hexanuclear, [Cu6(µ3-O)(HV)3(ClO4)7(H2O)9]·8H2O (1), one dodecanuclear, [Cu12(µ3-O)2(V)6(ClO4)5(H2O)18](ClO4)3·6H2O (2), and one tetradecanuclear 1D-polymer, {[Cu14(µ3-OH)2(V)6(HV)(ClO4)11(H2O)20](ClO4)2·14H2O} n (3), the last two containing hexanuclear subunits linked by perchlorato bridges. The Cu-Cu av. intra-triangle distance is 3.352(2) Å and the Cu(central)-Cu(bridged external) av. distance is 5.338(3) Å. The magnetic properties of the hexanuclear "Cu3O-3Cu" cluster have been studied, resulting as best fit parameters: g = 2.18(1), J(intra-triangle) = -247.0(1) cm-1 and j(central CuII - external CuII) = -5.15(2) cm-1.

Quantification of Vaporised Targeted Nanodroplets Using High-Frame-Rate Ultrasound and Optics.

Molecular targeted nanodroplets that can extravasate beyond the vascular space have great potential to improve tumor detection and characterisation. High-frame-rate ultrasound, on the other hand, is an emerging tool for imaging at a frame rate one to two orders of magnitude higher than those of existing ultrasound systems. In this study, we used high-frame-rate ultrasound combined with optics to study the acoustic response and size distribution of folate receptor (FR)-targeted versus non-targeted (NT)-nanodroplets in vitro with MDA-MB-231 breast cancer cells immediately after ultrasound activation. A flow velocity mapping technique, Stokes' theory and optical microscopy were used to estimate the size of both floating and attached vaporised nanodroplets immediately after activation. The floating vaporised nanodroplets were on average more than seven times larger than vaporised nanodroplets attached to the cells. The results also indicated that the acoustic signal of vaporised FR-targeted-nanodroplets persisted after activation, with 70% of the acoustic signals still present 1 s after activation, compared with the vaporised NT-nanodroplets, for which only 40% of the acoustic signal remained. The optical microscopic images revealed on average six times more vaporised FR-targeted-nanodroplets generated with a wider range of diameters (from 4 to 68 µm) that were still attached to the cells, compared with vaporised NT-nanodroplets (from 1 to 7 µm) with non-specific binding after activation. The mean size of attached vaporised FR-targeted-nanodroplets was on average about threefold larger than that of attached vaporised NT-nanodroplets. Taking advantage of high-frame-rate contrast-enhanced ultrasound and optical microscopy, this study offers an improved understanding of the vaporisation of the targeted nanodroplets in terms of their size and acoustic response in comparison with NT-nanodroplets. Such understanding would help in the design of optimised methodology for imaging and therapeutic applications.

3-D Microvascular Imaging Using High Frame Rate Ultrasound and ASAP Without Contrast Agents: Development and Initial In Vivo Evaluation on Nontumor and Tumor Models.

Three-dimensional imaging is valuable to noninvasively assess angiogenesis given the complex 3-D architecture of vascular networks. The emergence of high frame rate (HFR) ultrasound, which can produce thousands of images per second, has inspired novel signal processing techniques and their applications in structural and functional imaging of blood vessels. Although highly sensitive vascular mapping has been demonstrated using ultrafast Doppler, the detectability of microvasculature from the background noise may be hindered by the low signal-to-noise ratio (SNR) particularly in the deeper region and without the use of contrast agents. We have recently demonstrated a coherence-based technique, acoustic subaperture imaging (ASAP), for super-contrast vascular imaging and illustrated the contrast improvement using HFR contrast-enhanced ultrasound. In this work, we provide a feasibility study for microvascular imaging using ASAP without contrast agents, and extend its capability from 2-D to volumetric vascular mapping. Using an ultrasound research system and a preclinical probe, we demonstrated the improved visibility of microvascular mapping using ASAP in comparison to ultrafast power Doppler (PD) on a mouse kidney, liver, and tumor without contrast agent injection. The SNR of ASAP images improves in average by 10 dB when compared to PD. In addition, directional velocity mappings were also demonstrated by combining ASAP with the phase information extracted from lag-1 autocorrelation. The 3-D vascular and velocity mapping of the mouse kidney, liver, and tumor were demonstrated by stacking the ASAP images acquired using 2-D ultrasound imaging and a trigger-controlled linear translation stage. The 3-D results depicted clear microvasculature morphologies and functional information in terms of flow direction and velocity in two nontumor models and a tumor model. In conclusion, we have demonstrated a new 3-D in vivo ultrasound microvascular imaging technique with significantly improved SNR over existing ultrafast Doppler.

Development of 68Ga-labelled ultrasound microbubbles for whole-body PET imaging.

Microbubble (MB) contrast agents have revolutionalised the way ultrasound (US) imaging can be used clinically and pre-clinically. Contrast-enhanced US offers improvements in soft-tissue contrast, as well as the ability to visualise disease processes at the molecular level. However, its inability to provide in vivo whole-body imaging can hamper the development of new MB formulations. Herein, we describe a fast and efficient method for achieving 68Ga-labelling of MBs after a direct comparison of two different strategies. The optimised approach produces 68Ga-labelled MBs in good yields through the bioorthogonal inverse-electron-demand Diel-Alder reaction between a trans-cyclooctene-modified phospholipid and a new tetrazine-bearing HBED-CC chelator. The ability to noninvasively study the whole-body distribution of 68Ga-labelled MBs was demonstrated in vivo using positron emission tomography (PET). This method could be broadly applicable to other phospholipid-based formulations, providing accessible solutions for in vivo tracking of MBs.

Optically and acoustically triggerable sub-micron phase-change contrast agents for enhanced photoacoustic and ultrasound imaging.

We demonstrate a versatile phase-change sub-micron contrast agent providing three modes of contrast enhancement: 1) photoacoustic imaging contrast, 2) ultrasound contrast with optical activation, and 3) ultrasound contrast with acoustic activation. This agent, which we name 'Cy-droplet', has the following novel features. It comprises a highly volatile perfluorocarbon for easy versatile activation, and a near-infrared optically absorbing dye chosen to absorb light at a wavelength with good tissue penetration. It is manufactured via a 'microbubble condensation' method. The phase-transition of Cy-droplets can be optically triggered by pulsed-laser illumination, inducing photoacoustic signal and forming stable gas bubbles that are visible with echo-ultrasound in situ. Alternatively, Cy-droplets can be converted to microbubble contrast agents upon acoustic activation with clinical ultrasound. Potentially all modes offer extravascular contrast enhancement because of the sub-micron initial size. Such versatility of acoustic and optical 'triggerability' can potentially improve multi-modality imaging, molecularly targeted imaging and controlled drug release.

Design and validation of a new ratiometric intracellular pH imaging probe using lanthanide-doped upconverting nanoparticles.

pH homeostasis is strictly controlled at a subcellular level. A deregulation of the intra/extra/subcellular pH environment is associated with a number of diseases and as such, the monitoring of the pH state of cells and tissues is a valuable diagnostic tool. To date, only a few tools have been developed to measure the pH in living cells with the spatial resolution needed for intracellular imaging. Among the techniques available, only optical imaging offers enough resolution and biocompatibility to be proposed for subcellular pH monitoring. We present herein a ratiometric probe based on upconversion nanoparticles modified with a pH sensitive moiety for the quantitative imaging of pH at the subcellular level in living cells. This system provides the properties required for live cell quantitative imaging i.e. positive cellular uptake, biocompatibility, long wavelength excitation, sensitive response to pH within a biologically relevant range, and self-referenced signal.

Correction: Near infrared activation of an anticancer Pt(IV) complex by Tm-doped upconversion nanoparticles.

Correction for 'Near infrared activation of an anticancer Pt(IV) complex by Tm-doped upconversion nanoparticles' by Emmanuel Ruggiero et al., Chem. Commun., 2015, 51, 2091-2094.

Near infrared activation of an anticancer Pt(IV) complex by Tm-doped upconversion nanoparticles.

The Pt(IV) complex cis,cis,trans-[Pt(NH3)2(Cl)2(O2CCH2CH2CO2H)2] is photoactivated by near infrared light (980 nm) using NaYF4:Yb(3+)/Tm(3+)@NaYF4 core-shell upconversion nanoparticles. Coupling of this cisplatin precursor with the biocompatible PEGylated phospholipid DSPE-PEG(2000)-NH2 affords a valuable approach to decorate the surface of the nanoparticles, providing novel photoactivatable nanomaterials capable of releasing Pt(II) species upon NIR light excitation.

An Iron Oxide Nanocarrier Loaded with a Pt(IV) Prodrug and Immunostimulatory dsRNA for Combining Complementary Cancer Killing Effects.

There is major current interest in harnessing the immune system against cancer and in developing drugs that provide complementary cancer killing mechanisms. Although the recent advent of nanoparticle-based drug delivery systems has improved the efficacy of platinum drugs for chemotherapy, one of the fundamental paradigms in their design and use is evading surveillance by the immune system to enhance anticancer efficacy. However, new studies are showing that chemotherapy can profit from actively targeting stimulation of the immune system and that suitably functionalized nanomaterials might be ideal for overcoming some key challenges in immunotherapy. Pt(IV) prodrug-modified PEGylated phospholipid micelles that encapsulate biocompatible iron oxide nanoparticles (IONPs) as a new delivery system for cisplatin are reported. The Pt(IV)-IONPs are functionalized with polyinosinic-polycytidylic acid (poly (I:C))--a double stranded RNA (dsRNA) analog widely used as an adjuvant in clinical trials of cancer immunotherapy. The Pt(IV)-IONPs and poly (I:C)--Pt(IV)-IONPs enhance by more than an order of magnitude the prodrug cytotoxicity in different tumor cells, while greatly increasing the ability of cisplatin and poly (I:C) to activate dendritic cells--the key cellular players in immunotherapy. The results suggest that these constructs hold promise for targeted chemoimmunotherapy.